Project Summary Viral subunit vaccines are safe and convenient, but generally suffer low efficacy. Our overall hypothesis is that an intrinsic limitation is associated with subunit vaccine designs in which artificially exposed surfaces of subunit vaccines contain epitopes unfavorable for vaccine efficacy. The receptor-binding domain (RBD) of a coronavirus spike protein consists of a core subdomain that serves as the structural scaffold and a receptor- binding motif (RBM) that binds the receptor and contains neutralizing epitopes. The RBDs are prime candidates for subunit vaccine designs. In preliminary studies, we identified epitopes on the core subdomain of MERS coronavirus (MERS-CoV) RBD that were buried in the full-length spike protein but became artificially exposed in recombinant RBDs. We further showed that these epitopes severely reduce vaccine efficacy by inducing strong non-neutralizing immune responses and distracting the host immune system from reacting to the neutralizing epitopes on the RBM. This novel finding reveals an intrinsic limitation of viral subunit vaccines that the vaccine field had been unaware of. In this proposal, we aim to characterize this intrinsic limitation and establish novel approaches to overcome it. We use the RBDs from highly pathogenic coronaviruses, including MERS-CoV and SARS coronavirus (SARS-CoV), as the model system. This proposal contains three major design approaches for coronavirus RBD vaccines. First, we will identify and characterize the artificially exposed unfavorable epitopes on the core subdomain of coronavirus RBDs. To this end, we introduce a novel concept, neutralizing immunogenicity index (NII), to evaluate the contribution of each epitope to the overall vaccine efficacy. We will mask the negative epitopes on the core subdomain through glycan shielding or resurfacing. This design enhances the efficacy of the individually optimized RBD vaccines. Second, we will construct chimeric RBDs containing the core subdomain from one coronavirus RBD as the structural scaffold and the RBM from another coronavirus RBD as the immunogenic sites. The unfavorable epitopes on the core subdomain should have been silenced from the first design approach. The interface of the core subdomain and RBM will be optimized to maximize the stability of the chimeric RBD vaccines. This design prepares us for the emergence of highly pathogenic coronaviruses in the future. Third, we will construct nanoparticle-carried coronavirus RBD vaccines in a way that artificially exposed unfavorable epitopes on the core subdomain are re-buried at the molecular interfaces to enhance the RBD vaccine's efficacy. We will use mice to evaluate the immunogenicity of the above engineered RBD vaccines and will use animal models (including hDPP4-knock-in (KI)) mice and non-human primates) to assess the selected RBD vaccines against live coronavirus challenge. Overall, this research establishes the artificially exposed unfavorable epitopes as the intrinsic limitation of viral subunit vaccines and finds novel approaches to overcome it. Therefore, this research holds the promise of making subunit vaccines a more successful and widely used strategy in combating virus infections.